Methylation effect on chalcone synthase gene expression determines anthocyanin pigmentation in floral tissues of two Oncidium orchid cultivars

Abstract

The anthocyanin-biosynthetic pathway was studied in flowers of Oncidium Gower Ramsey with yellow floral color and mosaic red anthocyanin in lip crests, sepals and petals, and compared with the anthocyanin biosynthesis in flowers of Oncidium Honey Dollp, a natural somatoclone derived from tissue culture of Gower Ramsey, with a yellow perianth without red anthocyanins in floral tissues. HPLC analysis revealed that the red anthocyanin in lip crests of the Gower Ramsey cultivar comprised peonidin-3-O-glucoside, delphinidin-3-O-glucoside and cyanidin-3-O-glucoside, whereas Honey Dollp was devoid of anthocyanin compounds. Among the five anthocyanin-biosynthetic genes, OgCHS was actively expressed in lip crests of Gower Ramsey flowers, but no transcripts of OgCHS were detected in Honey Dollp floral tissues. Transient expression of OgCHS by bombardment confirmed that recovery of the OgCHS gene expression completed the anthocyanin pathway and produced anthocyanin compounds in lip crests of Honey Dollp flowers. Transcription factor genes regulating anthocyanin biosynthesis showed no distinctive differences in the expression level of OgMYB1, OgbHLH and OgWD40 between the two cultivars. A methylation assay revealed that the promoter of OgCHS was not methylated in Gower Ramsey, while a positive methylation effect was present in the upstream promoter region of OgCHS in Honey Dollp. Overall, our results suggest that the failure of anthocyanin accumulation in Honey Dollp floral tissues may be attributed to inactivation of the OgCHS gene resulting from the epigenetic methylation of 5'-upstream promoter region.

Transcriptome and Proteome Data Reveal Candidate Genes for Pollinator Attraction in Sexually Deceptive Orchids

“…The widespread presence of candidate gene transcripts in all species therefore suggests that species differences in pollinator attraction, and consequently reproductive isolation, are unlikely to be caused by the simple presence or absence of expression in any candidate gene class. Rather, it may be expected that subtler changes, such as paralogue-, isoform-, or allele-specific sequence and/or expression changes, or even epigenetic phenomena [114], [115], underlie reproductive isolation. This holds true at least for SAD genes specifying alkene double-bond positions [20], [24] and appears similarly plausible for KCS and hydrocarbon chain length, where the presence of 25 transcript sequences presages complexity.…”

Substantial Epigenetic Variation Causing Flower Color Chimerism in the Ornamental Tree Prunus mume Revealed by Single Base Resolution Methylome Detection and Transcriptome Sequencing

“…In previous studies, gene methylation has been observed to influence floral pigmentation. For instance, red pigmentation is observed in the flowers of the transgenic petunia line 17-R upon hypomethylation of the 35S promoter driving the A1 gene [26], while mosaic red anthocyanin in lip crests, sepals, and petals of yellow flowers of Oncidium “Gower Ramsey” may be attributed to activation of the OgCHS gene resulting from the demethylation of the five-upstream promoter region [27]. As a third example, methylation levels of MYB genes are associated with the formation of red-skinned pears and apples [37,38].…”

“…persica “Genpei” [25]. At the same time, epigenetic modification, such as the use of hypomethylated promoters of A1 , DFR - B , and OgCHS genes driving the brick-red pelargonidin pigmentation of flower tissue [11,26,27], has been introduced to reveal genetic variation in variegated flowers.…”

How to perform RT-qPCR accurately in plant species? A case study on flower colour gene expression in an azalea (Rhododendron simsii hybrids) mapping population

“…The exploration of natural flower colour differences by means of gene expression studies is only done between a limited number of genotypes, e.g. in cyclamen [19], Ipomoea [20], Freesia hybrida [21], azalea [22,23] or Oncidium [24]. No data are currently available on the consistent effect of the studied genes in other genotypes with the same flower colour.…”

“…Moreover, the quantification methods used in the aforementioned studies are not the most accurate. Some studies still describe the use of Northern blots [18,24] or semi-quantitative RT-PCR (reversed transcription PCR) [16,19,21,23,24], others do use quantitative RT-PCR (RT-qPCR) but limit themselves to the comparative Cq (quantification cycle) method [25] in combination with the use of only a single non-validated reference gene. However, multiple, assay-validated reference genes are considered to be an essential component of a consistent RT-qPCR assay [26-30].…”